Phosphoinositides are important mediators of hormone and growth factor signals that determine cell proliferation, survival, morphology, metabolism and motility. Phosphoinositide kinases, phosphatases and the proteins that they interact with are directly involved in diseases such as cancer and diabetes. Ptdlns-5-P is the most recently identified phosphoinositide in cells and its function is largely unknown. PIP4k II is a lipid kinase that uses Ptdlns-5-P as a substrate to synthesize Ptdlns-4,5-P2. This phosphoinositide pathway is referred to as the alternative pathway for Ptdlns-4,5-P2 synthesis because it only contributes to a fraction of the total Ptdlns-4,5-P2 in cells. The ultimate goal of this project is to understand the role of Ptdlns-5-P and the alternative pathway for Ptdlns-4,5-P2 synthesis in cell function. Several studies have indicated that the alternative pathway may be regulated by extracellular signals. Further, overexpression of PIP4k II in cells attenuates the signal initiated by insulin by activating the phosphoinositide phosphatase Ship2, while disruption of the PIP4k lib gene in mice increases sensitivity to insulin, a phenotype that resembles Ship2 disruption. The purpose of this study is to test the hypothesis that Ptdlns-5-P is a signaling molecule and that the PIP4k II function in cells is to regulate the levels of Ptdlns-5-P. The first step will be to identify the pathways that lead to Ptdlns-5-P synthesis in cells and to investigate how the levels of this lipid can be regulated by extracellular factors. For this purpose, the enzymes that synthesize Ptdlns-5-P in vitro will be tested for their ability to regulate Ptdlns-5-P in cells. The next step will be to determine which components of the alternative pathway are involved in ship2 activation. PIPk chimeras will be used to determine whether Ptdlns-5-P phosphorylation by PIP4k II is necessary and/or sufficient for Ship2 regulation. The possibility that Ptdlns-5-P is a negative regulator of Ship2 will be explored by testing whether Ptdlns-5-P by itself can regulate Ship2. The third step will be to elucidate the mechanism for Ship2 activation by PIP4k I1. Each domain and regulatory sites in the Ship2 protein will be mutated to assess their role in localization and activation mediated by PIP4k I1.The accomplishment of these goals will set the foundation for designing new strategies to intervene in the disease etiology involving phosphoinositide metabolism malfunction.